684 Chem. Commun., 2012, 48, 684–686 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Commun., 2012, 48, 684–686

A near-infrared fluorescent probe for monitoring ozone and imaging in

living cellsw

Kehua Xu, Shuxia Sun, Jing Li, Lu Li, Mingming Qiang and Bo Tang*

Received 21st September 2011, Accepted 8th November 2011

DOI: 10.1039/c1cc15844a

A near-infrared fluorescent probe (Trp-Cy) for endogenous

ozone is presented, which exhibited a large stokes shift about

140 nm and a rapid fluorescence response to ozone with high

selectivity and sensitivity.

The investigation of reactive oxygen species (ROS) in living

cells has accelerated the process of understanding the relation-

ship of oxidative stress to diseases, along with the development

of imaging technology. However, it is predestined that new

species of ROS generated from living cells will be constantly

discovered due to the complexity of cells. For example,

Wentworth and associates recently observed that antibodies can

catalyze the generation of previously unknown oxidants, including

dihydrogen trioxide (H2O3) and ozone (O3) in neutrophils

(PMNs) isolated from human peripheral blood,1 and reported

on ozone formation in human atherosclerotic arteries,2 although

ozone (O3) as an oxidizer harmful to human health has been

widely studied.3,4 Zhang et al. subsequently found that the

cholesterol ozonolysis products existed in clinical brain samples

and suggested that they can likely trigger misfolding of protein in

sporadic amyloid disease.5 Thus, we think that a fascinating area

of study on O3 has been opened up, while at the same time we also

realize that the lack of ozone probes suitable for monitoring O3 in

living cells will suppress its development.

Indigo carmine is a sensitive probe for O3 detection in

aqueous systems,6 but it is not a specific one.1 Recently,

Garner and coworkers synthesized a fluorescent molecular

probe (lex/lem = 497/523 nm) to successfully detect ozone in

both biological and atmospheric samples and visualize the

level of O3 in human bronchial epithelial cells when these cells

were exposed to ozone.7 However, to the best of our knowledge,

rapid and specific methods for the detection of endogenous

ozone have not yet been reported.

Ozone is a highly reactive oxygen species and has a very

short half-life of 66 s and very low concentration in living cells.

Thus, in order to effectively detect ozone derived from cells, a

new probe should be developed. Near-infrared (NIR) fluo-

rescent probe, as is well-known, is an excellent sensor for

biomolecules, being capable of affording high spatial resolution

and deep tissue imaging, and of minimizing tissue auto-

fluorescence.8 Therefore, our strategy of probe design was to

choose tricarbocyanine (Cy), a near-infrared (NIR) fluorescent

dye with a high extinction coefficient,9–11 as a signal transducer

and to choose L-tryptophan (Trp), as an O3-indicator12 to

synthesize a new optical probe, Trp-Cy, for O3 detection

(Scheme 1), based on a twisted intramolecular charge transfer

(TICT) mechanism (see ESI, Fig. S1w).13 The synthesized

probe was successfully applied for the first time to detecting

and imaging O3 derived from living cells.

Trp-Cy was easily synthesized through a simple one-step reaction

and was purified by column chromatography. The structure of

Trp-Cy was characterized with 1H-NMR, 13C-NMR and MS

(see ESIw). The spectral properties of Trp-Cy and Cy were

compared, as shown in Fig. 1a. Trp-Cy has a larger stokes shift

about 140 nm and its lmax of fluorescence excitation and emission

lies at 630 nm and 770 nm, respectively, which is a very desired

property in probe design because it can improve the detection

sensitivity and reduce photobleaching. In addition, the oxidized

Trp-Cy from the reaction of Trp-Cy with O3 was characterized by

MS (see ESIw).The response of Trp-Cy to O3 was tested and the optimal

conditions were determined (Figs S2–S4). As expected, upon

the reaction with different concentrations of O3, a gradual

increase in the fluorescence intensity was observed in Fig. 1b

and there was a good linearity between the relative fluores-

cence intensity (DF) and O3 concentration in the range

0.05–7.0 mM. The regression equation was DF = 654.3 +

1784.8 � [O3] mM with a linear coefficient of 0.9953. The limit

Scheme 1 The synthesis of Trp-Cy and reaction of Trp-Cy with O3.

Key Laboratory of Molecular and Nano Probes, Engineering ResearchCenter of Pesticide and Medicine Intermediate Clean Production,Ministry of Education, College of Chemical Engineering andMaterials Science, Shandong Normal University, Jinan, 250014,China. E-mail: tangb@sdnu.edu.cnw Electronic supplementary information (ESI) available: Details ofsynthesis, characterization, fluorescence properties and imaging ofTrp-Cy. See DOI: 10.1039/c1cc15844a

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 684–686 685

of detection was 17 nM (standard deviation 10.1, n = 11).

Furthermore, a fast response of Trp-Cy to O3 was confirmed

by a kinetics experiment, in which the fluorescence intensity

instantly increased by adding O3 into the probe solution and

the intensity was kept level for at least 25 min, as shown in

Fig. 2a.

The selectivity of Try-Cy was studied. Considering the

complexity of the intracellular environment, the interferences

of various bioanalytes were carried out, including other reactive

oxygen species (ROS), reactive nitrogen species (RNS) and

biological antioxidants, such as glutathione (GSH) and ascorbic

acid (Vc). Also, in consideration that L-tryptophan could bind

many metal ions in solution, an additional test was performed

in order to determine whether metal ions, such as K+,

Na+,Ca2+, Mg2+, Zn2+, Cd2+, Co2+, Ni2+ and Cu2+, are

potential interferences. The tolerance level was defined as a

relative error not exceeding �5% in the determination of the

analytes. The results showed that the Trp-Cy probe possessed

high selectivity towards O3, as shown in Figs 2b and S5aw. Totake into account the application of Trp-Cy in cell imaging, an

experiment on extending the reaction time to 30 min was

performed. Fig. S5b showed that these interferences did not

affect the detection of O3. In addition, the molecular mechanism

of this probe to specifically recognize ozone was discussed using

mass spectrometry (see Fig. S6 and Scheme S1w).With the above results, the application of Trp-Cy was firstly

operated in living A549 lung cancer cells because lung cells are

susceptible to ozone. The A549 cells were incubated with 10 mMTrp-Cy for 30 min at 37 1C; no obvious fluorescence appeared,

as shown in Fig. 3a. When the above cells were supplemented

with 6 mM O3 for 10 min, as expected, a bright fluorescence

was observed, as shown in Fig. 3b. The bright-field image of

(b) is shown in Fig. 3c. In order to confirm the selectivity of

Trp-Cy to ozone in living cells, probe-loaded cells were

incubated with SNP (NO donor), NaClO and O2�� generated

by X/XO, as illustrated in Figs 3d–f. The experimental results

displayed that the probe can selectively respond to the change

of O3 levels in living cells.

Secondly, we chose the mouse macrophage cell line

RAW264.7 to monitor endogenous ozone, since macrophage

cells can activate the generation of ozone after exposure to

phorbol 12-myristate 13-acetate (PMA).14 RAW264.7 cells

were stimulated by PMA, and then incubated with 10 mMTrp-Cy for 30 min. There was almost no fluorescence in the

absence of stimulant in Fig. 4a, while strong fluorescence

appeared after treatment with PMA (100 ng mL�1) for 20 min,

as shown in Fig. 4b. When the cells were pre-treated with ethyl

4-vinylbenzoate (0.1 mM) as a specific scavenger of ozone,15

much weak fluorescence similar to the control panel was

observed in the stimulated cells, as shown in Fig. 4c. The

experiment results illustrated that the strong fluorescence in

Fig. 4b was indeed induced by ozone, rather than other ROS.

We then applied Trp-Cy to probe the subcellular locations

of endogenous ozone in RAW264.7 cells using confocal

fluorescence microscopy. PMA-stimulated RAW 264.7 cells

were incubated with 10 mM Trp-Cy for 10 min and then

incubated with a fluorescent nuclear stain (DAPI, 10 mg mL�1)

for 10 min. Fluorescence images were obtained, as shown in

Figs 4d–f. Co-staining with DAPI revealed the location of the

probe in the cytoplasm of RAW264.7 cells. Furthermore, con-

sidering that cyanine dyes16 can be selectively accumulated by the

mitochondria of living cells, a mitochondrial-targeted experiment

using the synthesized probe was supplemented. PMA-stimulated

RAW 264.7 cells were incubated with 10 mM Trp-Cy for 10 min

and then incubated with 50 nMMito Tracker Green FM (a green-

fluorescent mitochondrial stain) for 10 min. The fluorescence

Fig. 1 (a) Fluorescent spectra of 10 mM Cy and 10 mM Trp-Cy in

PBS at pH 7.4. (b) Fluorescence responses of 10 mM Trp-Cy to

different concentrations of O3. Inset: A linear correlation between

emission intensities and concentrations of O3 (lex/lem = 630/770 nm).

Fig. 2 (a) The time course of the fluorescence intensity of 10 mMTrp-Cy

to O3 (0 mM and 6 mM) in PBS at pH 7.4, measured at 770 nm with

excitation at 630 nm. (b) The relative fluorescence responses of Trp-Cy to

ROS, RNS and biological antioxidants (6 mM for O3 and ONOO�; 12 mMfor NO; 24 mM for 1O2 and NaClO; 30 mM for �OH, t-BuOOH; 120 mMfor H2O2 and O2

��; 0.3 mM for GSH and Vc). Black bars represent the

addition of one of these interferences to a 10 mM solution of Trp-Cy. Gray

bars represent the addition of O3 or O3 plus one of these interferences to

the probe solution. All data were acquired in 30 mM phosphate buffer

with pH 7.4 at 37 1C (lex/lem = 630/770 nm).

Fig. 3 Confocal fluorescence images of living A549 cells using a 633 nm

laser. (a) A549 cells incubated with 10 mMTrp-Cy at 37 1C for 30 min. (b)

Probe-loaded cells incubated with 6 mM O3 for 10 min. (c) A bright-field

image of (b). (d–f) Probe-loaded cells incubated with NO, NaClO and

O2�� (6 mM for each) for 10 min. All cells were rinsed three times with

0.1 M PBS buffer at room temperature before imaging.

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686 Chem. Commun., 2012, 48, 684–686 This journal is c The Royal Society of Chemistry 2012

images were obtained, as shown in Figs 5a–c. The overlay image

displayed that Trp-Cy is indeed a mitochondrial-targeted

O3 probe.

Finally, the cytotoxicity of Trp-Cy and photostability of

oxidized Trp-Cy were investigated. A methyl thiazolyl tetra-

zolium (MTT) assay was first performed in A549 cells (5 � 104

cell mL�1) dispersed within replicate 96-well microtiter plates

with probe concentrations of 10–500 mM. Absorbance was

measured at 490 nm in a TRITURUS microplate reader, as

shown in Fig. S7w. The result showed the cell viability was

90% under the experimental conditions. Then a photo-bleaching

test was carried out by means of time-sequential scanning of

the probe-loaded A549 cells incubated with 6 mMO3 for 10 min.

After 300 s of continuous irradiation with a 633 nm laser, no

obvious changes were observed in fluorescence brightness of

oxidized Trp-Cy (Figs S8 and S9w).In summary, we developed a novel NIR fluorescent probe

(Trp-Cy) for ozone. The synthesized probe has a large stokes

shift about 140 nm and possesses low cytotoxicity. The

response of Trp-Cy to O3 was rapid, highly sensitive and

selective, and the fluorescence imaging of O3 in living cells

was successfully achieved using confocal laser scanning micro-

scopy. Our probe should provide valuable tools with which to

better understand ozone’s role in human health.

This work was supported by the National Key Natural

Science Foundation of China (No. 21035003), National Natural

Science Funds for Distinguished Young Scholar (No.20725518),

National Natural Science Foundation of China (No.20875057),

Key Natural Science Foundation of Shandong Province of

China (Nos. ZR2010BZ001 and ZR2011BZ006), the Science

and Technology Development Programs of Shandong Province

of China (No. 2008GG30003012), and Program for Changjiang

Scholars and Innovative Research Team in University.

Notes and references

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2 P. Wentworth Jr., J. Nieva, C. Takeuchi, R. Galve,A. D. Wentworth, R. B. Dilley, G. A. DeLaria, A. Saven,B. M. Babior, K. D. Janda, A. Eschenmoser and R. A. Lerner,Science, 2003, 302, 1053.

3 G. C. Chuang, Z. Yang, D. G. Westbrook, M. Pompilius,C. A. Ballinger, C. Roger White, D. M. Krzywanski,E. M. Postlethwait and S. W. Ballinger, Am. J. Physiol.: LungCell. Mol. Phys., 2009, 297, 209.

4 S. A. Jorge, C. F. Menck, H. Sies, M. R. Osborne, D. H. Phillips,A. Sarasin and A. Stary, DNA Repair, 2002, 1(5), 369.

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6 K. Takeuchi and I. Takeuchi, Anal. Chem., 1989, 61, 619.7 A. L. Garner, M. S. C. Claudette, R. P. Bruce, D. L. George,A. Shin and K. Kazunori, Nat. Chem., 2009, 1, 316.

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10 K. H. Xu, L. L. Wang, M. M. Qiang, L. Y. Wang, P. Li andB. Tang, Chem. Commun., 2011, 47, 7386.

11 K. H. Xu, H. C. Chen, J. W. Tian, B. Y. Ding, Y. X. Xie,M. M. Qiang and B. Tang, Chem. Commun., 2011, 47, 9468.

12 W. A. Pryor and R. M. Uppu, J. Biol. Chem., 1993, 268, 3120.13 R. G. Zbigniew and R. Krystyna, Chem. Rev., 2003, 103, 3899.14 A. J. Kettle, B. M. Clark and C. C. Winterbourn, J. Biol. Chem.,

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Fig. 4 Confocal fluorescence images of living RAW 264.7 cells. (a)

Cells incubated with 10 mM Trp-Cy at 37 1C for 30 min. (b) Cells

incubated with 100 ng mL�1 PMA for 20 min and then incubated with

10 mM Trp-Cy at 37 1C for 30 min. (c) Cells incubated with 0.1 mM

ethyl 4-vinylbenzoate and then treated according to (b). (d) PMA-

stimulated cells incubated with 10 mM Trp-Cy at 37 1C for 10 min and

then incubated with DAPI (10 mg mL�1) for 10 min, using a 633 nm

laser. (e) The image of the (d) cells using a 405 nm laser. (f) One

overlay image of (d) and (e). All cells were rinsed three times with

0.1 M PBS buffer at room temperature before imaging.

Fig. 5 Confocal fluorescence images of living mice macrophages

(RAW 264.7). (a) PMA-stimulated cells incubated with 10 mMTrp-Cy at 37 1C for 10 min and then incubated with 50 nM Mito

Tracker Green FM for 10 min, using a 633 nm He–Ne laser. (b) The

above cells were excited by a 488 nm argon laser. (c) One overlay

image of (a) and (b). All cells were rinsed three times with 0.1 M PBS

buffer at room temperature before imaging.

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